PROJECT SUMMARY The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 10 million people in the Americas, with an at-risk population of 70 million. While the acute infection by the parasite is effectively controlled by the immune system, a chronic infection can persist for the lifetime of the host. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease remains underreported, understudied and underfunded. Basic research into the biology of T. cruzi has been previously hindered by a lack of efficient genetic tools, but the advent of CRISPR/Cas9 gene editing technology has cleared the way for more in-depth molecular analyses. The primary interface between T. cruzi and its mammalian hosts is at the level of the replicative intracellular amastigotes in the cytoplasm of infected host cells. Trypanosoma cruzi is one of only a few protozoan pathogens that live and replicate directly in the cytosol of nucleated cells. Of the (mostly bacterial) pathogens that can inhabit the host cytosol, many have been shown to actively induce host cytosolic protein degradation in order to obtain energy and amino acids which are normally limiting in the cytoplasm. A long-standing mystery is how T. cruzi is able to extract necessary nutrients from this impoverished environment? Unlike other parasites, T. cruzi has the ability to endocytose and digest host cytosolic material via a long tubular invagination (cytopharynx) starting at a surface plasma membrane pore (cytostome). This structure, referred to here as the cytostome/cytopharynx or CSP complex, is present only in replicating forms of the T. cruzi and disassembles during the transition to trypomastigote stages. The CSP structure, which has been examined extensively using electron tomography techniques, has resisted molecular analysis, as the protein components comprising it have remained elusive. We have recently identified the first protein targeted solely to the CSP structure, CP1, and our preliminary work has shown that this protein colocalizes with endocytosed cargo in the CSP. In this proposal, we will use CP1 to carry out the first in-depth characterization of the T. cruzi CSP. We will fuse CP1 to the BioID biotin ligase and perform proximity labeling of the CSP to identify its protein components. A selected subset of the identified components will be validated by endogenous tagging and assessed for their role in parasite feeding, replication and survival. Additionally, we will extend our analysis of the host cell components taken up in the CSP to determine the selectively with respect to host proteins or organelles during intracellular amastigote replication. Completion of this study will allow us to begin analyzing in detail a unique, but crucial, aspect of T. cruzi parasitism which previously resisted in-depth investigation.